Derivative compounds of N-6-Trimethy-L-Lysine for therapeutic use

ABSTRACT

The invention provides derivative compounds of N-6-trimethyl-L-lysine (TML) for potential treatment of disorders resulting from deficiencies in the TML-carnitine pathway. The invention also provides a method of purification of TML and TML derivative compounds. The treatment of conditions of the diseases late infantile neuronal ceroid lipofuscinosis (LINCL) and neuronal ceroid lipofuscinosis (NCL) with TML were shown in the original parent application.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 11/105,165, filed Apr. 13,2005, which claims priority from provisional application No. 60/601,095filed on Aug. 12, 2004, and incorporates the subject matter identifiedas Inventions II and IV in the Requirement for Restriction/Election ofApr. 13, 2007, in the parent application. All materials referenced inthe prior provisional and non-provisional applications are herebyincorporated by reference. This includes, but is not limited to, allspecifications, drawings, and like materials.

Related divisional applications claiming similar priority include“Method of Synthesis and Purification of N-6-Trimethyl-L-Lysine andDerivative Compounds,” Attorney Docket Attorney Docket No. ChG_(—)00111;“Method of Treating Human Being for a Class of Metabolic Defects andEnergy Production Disorders,” Attorney Docket No. ChG_(—)00113; and“Method of Treating a Human Being for a Class of Neurological Defectsand Seizure Disorders,” Attorney Docket No. ChG_(—)00114.

All books, manuals, articles, and papers that are cited herein arehereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to the derivative compounds synthesized fromN-6-trimethyl-L-lysine (TML), which may be used in the treatment ofdeficiencies in the TML-carnitine biochemical pathway.

2. Description of Related Art

All material referenced in the prior provisional and non-provisionalapplications are hereby incorporated by reference.

In the parent application Ser. No. 11/105,165, allowed on Nov. 17, 2010to issue as a patent, it was shown by carefully compiled experimentalresults and other scientific demonstration, that therapeutic use of TMLmay successfully arrest, and in certain respects, reverse thedegeneration associated with the group of progressive neurologicaldiseases called neuronal ceroid lipofuscinoses (NCL).

The modified TML derivatives described should have similar ornear-similar results and improved biochemical properties. One ofordinary skill in the art would recognize that structural derivatives ofTML, such as those mentioned in this application, may participate in thesame biological processes and have the same and improved biochemicalproperties.

Formulations or encapsulations of the compounds shown in Formulas I-VImay be used for efficient intracellular delivery and as a prodrug of TMLto proceed to make endogeneous L-carnitine, and to participate invarious metabolic activities in the intermediate steps of L-carnitinebiosynthesis pathway. These may be used for better adsorption ofmodified TML into various tissues such as kidney, liver and brain. TheR′, R″, and aminoacyl groups are expected to hydrolyze inside thecellular media with one or more intracellular esterases to release freeTML. Intracellular esterases are known to hydrolyze esters (Ghosh, M.and Mitra, A. K., Effects of 5′-Ester Modification on thePhysicochemical Properties and Plasma Protein Binding of5-Iodo-2′-Deoxyuridine. Pharm. Res., 8, 771-775, 1991).

The parent application described the symptoms and common as well asdistinct characteristics of the spectrum of NCL group, including thefollowing: Batten Disease, Santavuori disease, Late-Infantile NeuronalCeroid Lipofuscinoses (LINCL), Speilmeyer-Sjogren disease, Kuf disease,Parry disease, Bernheimer-Seitelberger syndrome, Bielschowsky amauroticidiocy, Bielschowsky disease, Jansky-Bielschowsky disease, Seitelbergerdisease, late infantile amaurotic idiocy, late infantile Batten disease,subacute late infantile neuronal ceroid-lipofuscinosis,Zeman-Dyken-Lake-Santavuori-Savukoski disease.

At the genetic level, the neuronal ceroid lipofuscinoses (NCL's) resultfrom mutations in at least eight genes, and these mutations areresponsible for causing the various expressions of the neurodegenerativediseases collectively identified as NCLs. A summary background of thesemutations and a survey of the background reference literature were givenin the parent application. See Table A by Gene Locus.

TABLE A Neuronal Ceroid Lipofuscinosis-Summary of Symptoms CLN1(Infantile) CLN2 (Late Infantile) CLN3 (Juvenile) CLN4a (Kufs Disease)CLN5 (Late Infantile, Finnish Variant) CLN6 (Late Infantile, Variant,Included, Variable age at onset) CLN7 CLN8 CLN8 (Northern EpilepsyVariant) CLN9 CLN 10 (Cathepsin D-Deficient, Congenital) SYMPTOMS CLN1CLN2 CLN3 CLN4 CLN5 CLN6 CLN7 CLN8 CLN9 CLN10 Dementia Yes Yes Yes YesYes Yes Seizures Yes Yes Yes Yes Yes Yes Yes Yes Yes yes (hyperkneticmovements, hand/feet tremors) Progressive Visual Yes Yes Yes Yes Yes YesYes No Yes newborn Failure infant Mental Retardation Yes Yes Yes Yes YesYes Yes Yes Loss Of Speech Yes Yes Yes Yes Yes Yes yes Regression of YesYes Yes Yes Yes yes Motor Development Ataxia Yes Yes Yes Yes Yes Yes yesMuscular Yes Yes Yes Yes yes Hypotonia/Dystonia Microcephaly Yes OpticAtrophy/ Yes Yes Yes Yes Macular Degeneration Retinitis PigmentosaMyoclonus Yes Yes Yes Yes Yes Yes No Cerebellar Atrophy Yes Yes Yes Yesyes Quadraparesis Yes Refractory Epilepsy Yes Behavioral Yes YesInvolvement (Anger Outburst, Physical Violence) Table A Notes: 1.According to Mole et al.. 2005, the clinical course of the NCL's includeprogressive dementia, seizures, and progressive visual failure (Fulltext available athttp://www.springerlink.com/content/xu2406100j81034w/fulltext.pdf). 2.Obviously, a ‘yes’ means that the symptom is a characteristic of thedisease. A ‘NO’ means that the OMIM synopsis from clearly stated thatthe specific symptom is NOT characteristic of that particular NCL. Anempty space for a particular symptom does not necessarily preclude itfrom being part of the characteristics of that particular NCL; it wasnot mentioned specifically in the OMIM synopsis. For instance, CLN6 isan LINCL (CLN2) variant. It did not specifically mention MentalRetardation or Loss of Speech or Cerebellar Atrophy; but it would behard to believe that Mental Retardation/Loss of Speech/Cerebellar wouldnot be part of the continuum. 3. References CLN1http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256730 CLN2http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204500 CLN3http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204200 CLN4ahttp://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204300 CLN5http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256731 CLN6http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=601780 CLN7http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610951 CLN8http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=600143 CLN8 (northernepilepsy variant)http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610003 CLN9http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=609055 CLN10http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610127

No generally effective treatment for many of the diseases mentionedabove is currently available and these diseases are generally fatal. Theline of treatment proposed in the parent application, is based onadministering in the form of a therapeutic agent of very high purity,any of the following to a human being in need thereof: (a)N-6-trimethyl-L-lysine, (b) a prodrug thereof, or (c) a pharmaceuticallyacceptable salt of said N-6-trimethyl-L-lysine or said prodrug.

The one common characteristics in all these disorders has been found tobe the accumulation of autofluorescent storage material in all tissues,particularly pronounced in the central nervous system. Thischaracteristic has been tied to the fundamental role of L-carnitine inmetabolism, such as the prevention of hyperammonimia, lipid peroxidationand fatty acid metabolism. It has also been found that availability ofTML as the rate limiting step in the regulation of feedback inhibitionfor L-carnitine biosynthesis is crucial to the biosynthesis ofL-carnitine. (Schematic 1). (F. M. Vaz and R. J. A. Wandars, Biochem.J., 361, 417-429, 2002).

A summary of the role of carnitine, which was described in greaterdetail in the parent application, is outlined below:

(a) From a biochemical standpoint, L-carnitine plays an essential rolein energy metabolism. In fatty acid metabolism, it serves as shuttlebetween the cell sap and the mitochondria inner-workings permittingbreakdown of the long carbon fragment. A major part of that role is inmaintaining a balance between the concentration of a compound calledacyl CoA in the cell compartments and in sugar metabolism.

Optimal ATP production from either dietary or stored fatty acids isdependent on L-carnitine. L-Carnitine has several roles, most of whichinvolve conjugation of acyl residues to the b-hydroxyl group of theL-carnitine with subsequent translocation of this complex from onecellular compartment to another.

(b) Defects in fatty acid oxidation are a source of major morbidity andare potentially rapidly fatal. Fatty acid oxidation defects encompass aspectrum of clinical disorders, including recurrent hypoglycemic,hypoketotic encephalopathy or Reye-like syndrome in infancy withsecondary seizures and potential developmental delay, progressive lipidstorage myopathy, recurrent myoglobinuria, neuropathy, and progressivecardiomyopathy.

(c) Administration of L-carnitine prevents acute ammonia toxicity andenhances the efficacy of ammonia elimination as urea and glutamine. Inaddition the cytotoxic effects of ammonia, possibly arising from lipidperoxidation, are ameliorated by L-carnitine. These data indicate thefeasibility of utilization of L-carnitine in the therapy of humanhyperammonemic syndromes.

(d) L-Carnitine deficiency can be defined as a decrease of intracellularL-carnitine is a factor in inhibition of the mitochondrial oxidation oflong-chain fatty acids during fasting causes heart or liver failure(which may cause encephalopathy by hypoketonaemia, hypoglycaemia andhyper-ammonium), lower acetylcholine synthesis in the nervous system.

L-carnitine plays a key and critical role in enhancing fat metabolism.Reports attest to the fact that L-carnitine works by transporting fattyacids to be burned for fuel, increasing both energy supply and leanmuscle mass. Most found that unless an individual is deficient inL-carnitine, it is an unnecessary ergogenic aid. This contrasts with anapparent need in case of L-carnitine deficiency (e.g., in the casepursued by the inventors of Late Infantile Neuronal CeroidLipofuscinosis—one form of Batten Disease), of the correct operation ofthe endogenous production of L-carnitine. This need was corroborated inthe observations of dogs with Batten Disease given exogenous L-Carnitine(Siakotos A. N., Hutchins G. D., Farlow M. R., Katz M. L., EuropeanJournal of Paediatric Neurology 5 Suppl A: 151-6, 2001) and those of theparents of the child who was afflicted with LINCL (discussed below). Shewas given exogenous L-carnitine for over three years without significantmetabolic changes or marked outward observations of her condition. Itwas only the delivery of exogenous TML to the afflicted LINCL child thatyielded significant metabolic and outward, observable changes to hercondition.

A discussion of the experimental results presented in the parentapplication showed effectiveness of TML therapy in the amelioration ofseveral symptoms including: hyperammonemia, a glutamine levels,insomnia, “nervousness” or myoclonus.

L-Carnitine may be essential or “conditionally” essential for severalgroups of people including: normal infants, premature infants, and bothchildren and adults suffering from a variety of genetic, infectious, andinjury-related illnesses. For example, some cardiomyopathies whichafflict children are due to metabolic errors or deficiencies. There isdata that supports treatment of some myocardial dysfunctions withL-carnitine supplementation. (Winter, S., Jue, K., Prochazka J.,Francis, P., Hamilton, W., Linn, L., Helton, E. (1995) J. Child Neurol.10, Supple 2: S45-51.)

For these and other reasons, all of which were described in detail inthe parent application, it is believed that TML, or its derivativeswhich had been proposed in the parent application, can be used for thetreatment of a human being diagnosed with one or more of the following:defects in carnitine biosynthesis pathway, inefficiency of endogeneousTML, over-accumulation of TML bound protein at the cellular level, renalfailure conditions, hyperammonemic Encepalophathy, over-accumulation ofglutamine in the brain, reduced and deficient fatty acid metabolism andshuttling of fatty acid in to mitochondria, insufficient ATP productionor subsequent energy production and all the cellular activitiesassociated with this events, defective fatty acid oxidation resultingfrom carnitine deficiency, hypoglycemia, hypoketotic, encephalopathy,Reye-like syndrome, for recurrent seizures and developmental delay, AIDSor AIDS-like conditions, over-accumulation of lipids causing myopathy,myoglobinuria, neuropathy, cardiomyopathy, ammonia over-production,hyperammonemic syndromes, over accumulation of triacylglycrols, Battendiseases, infantile neuronal lipofuscinoses diseases (Santavvoridiseases), Late infantile neuronal lipofuscinoses diseases(Jansky-Bielscowsky), Speilmeyer disease, Sjorgsen disease, Kufdiseases, Parry diseases, Juvenile or adult neuronal lipofuscinosesdiseases (“NCL”) disease, lysosomal accumulation of mitochondrial ATPsynthase subunit and their by products, ataxia and seizures, variousstages of mental impairment, (e.g., learning disability, clumsiness,stumbling, impaired motor skills, and dementia, hyperandrogenism causedby NCL, defective dopamine receptors caused by NCL, epileptic fits,myoclonic epilepsy, Parkinson's disease, and Alzheimer's disease.

In all the conditions as described above, TML derivatives would beequally or more effective after hydrolysis in cellular environment andliberation of free TML.

The experimental results presented during the prosecution of the parentapplication showed that the progress observed by the medical care-giversto the LINCL child-patient correlated with the administration of highpurity TML. See Table B below.

TABLE B Results After TML Therapy. Test Name Nov. 19, 2003 ClinicalRange Jan. 29, 2004 Clinical Range Hgb 14.4 high 14 normal HCT 42 high40.3 normal RDW 11.5 high 12.3 normal ABS Lymphocytes 2.2 low 2.5 normalGlycine 50 high 25 normal Taurine 24 high 19 normal Carnitine, Total 40normal 43 normal Carnitine, Esthers 7 normal 10 normal Alanine 87 high47 normal Carbon Dioxide 32 high 22 normal BUN 2 low 5 (low) (6 isnorm!) AST 60 high  50 (high) (40 is norm) Platelets 586 high 461 (high)(369 norm) Glutamine 99 high 70 normal Notes to the Table B: (a)HCB =hemoglobin, HCT = Hematocrit, RDW Red Cell Distribution Width, ABSabsolute, BUN Blood Urea Nitrogen, AST = Aspartate Amonotransferase)(b)The examining physicians comments of Nov. 19, 2003 regarding Table B:(I) Alanine is elevated, this may be seen in states with increasedpyruvate, (ii) Glutamine is increased, this may be seen, withHyperammonemia.; Clinical correlation is indicated. (c)The examiningphysicians comments on Jan. 29, 2004 that no significant elevation ofserum amino acid was seen. (d)The patient's glucose and potassiumincreased (Glucose 93 baseline to 132; Potassium 4.4 baseline to 4.8).Even though the follow up blood work was done after an all night fast,we did give her some “Gatorade” to drink before the blood test. This wasgiven with her Klonopin to wash it down and certainly could be acontributing factor to the rise in glucose and potassium.

It was also found during the administration of TML to the child that itwas essential that high, therapeutic-grade TML be used for treatmentpurposes.

Therefore, TML derivate compounds were invented and purified by the teamof current inventors. These inventions are listed in the presentapplication.

SUMMARY OF THE INVENTION

The present invention provides derivative compounds of the biologicallyactive TML compound, which may be used to treat various diseasesresulting from deficiencies in the TML-carnitine pathway, such as LateInfantile Neuronal Lipofuscinosis (LINCL) or Neuronal CeroidLipofuscinosis (NCL).

It is also believed these can be used to treat a human being diagnosedwith one or more of the following: defects in carnitine biosynthesispathway, efficiency of endogeneous TML, over-accumulation of TML boundprotein at the cellular level, reduced and deficient fatty acidmetabolism and shuttling of fatty acid in to mitochondria, insufficientATP production or subsequent energy production and all the cellularactivities associated with this events, defective fatty acid oxidationresulting from carnitine deficiency, hypoglycemia, hypoketotic, overaccumulation of triacylglycrols, lysosomal accumulation of mitochondrialATP synthase subunit and their by-products. This is described in detailin the allowed parent patent application Ser. No. 11/105,165.

In one embodiment, the invention provides a compound represented byFormula II:

wherein R′ is selected from the group consisting of an alkyl havingbetween 1 and 5 carbon atoms and an aromatic ring.

In one embodiment, the invention provides a compound represented byFormula III:

wherein R″ is an alkyl having 1 to 5 carbon atoms or CH3.

In one embodiment, the invention provides a compound represented byFormula IV:

wherein R″ is an alkyl having between 1 and 5 carbon atoms, or CH3 andR′ is an alkyl having between 1 and 5 carbon atoms or an aromatic ring.

In one embodiment, the invention provides a compound represented byFormula V:

wherein a, a′, b, b′; c, c′, d, d′, e, and e′ are independently selectedfrom H, deuterium, and an alkyl having between 1 and 5 carbon atoms; R′is selected from the group consisting of H, an alkyl having between 1and 5 carbon atoms and an aromatic ring; and, and each N isindependently selected from nitrogen and N15 labeled nitrogen.

In one embodiment, the invention provides a compound represented byFormula VI:

wherein the a, b, b′, c, c′, d, d′, e, and e′ are independently selectedfrom H, deuterium, and an alkyl having from 1 to 5 carbon atoms, andeach N is independently selected from nitrogen and N15 labeled nitrogen.

In another embodiment, the invention provides a method of purifying theTML compound represented by Formula I

to at least 98% purity.

In another embodiment, the invention provides a method of purifying theTML derivate compounds represented by Formulas II, III, IV, V, and VIabove to at least 98% purity.

The method of purifying the TML and TML derivative compounds involvesthe following steps:

-   -   1. running the TML compound or TML derivative through an ion        exchange resin column;    -   2. washing the ion exchange resin column with at least 4 times        the volume of water as the amount of present TML compound or TML        derivative;    -   3. eluting the washed TML compound or TML derivative from the        ion exchange resin column to obtain eluted TML or TML        derivative; and    -   4. triturating the eluted TML or TML derivative.

In a preferred embodiment, the method further involves:

-   -   5. dissolving the triturated TML into a polar solvent;    -   6. filtering the dissolved TML through a microglass membrane        filter; and    -   7. lyophilizing the filtered TML at room temperature.

Although the process was outlined in the original application and isoutlined in preceding paragraphs, a detailed summary of the generalprocess is outlined in the Detailed Description. The synthesis was animprovement of the method reported in the literature, Frederic M. Vaz,Bela Melegh, Judith Bene, Dean Cuebas, Douglas A. Gage, Albert Bootsma,Peter Vreken, Albert H. van Gennip, Loran L. Bieber and Arnold J. A.Wanders; Clin. Chem.: 48:6, 826-834, 2002), and in the application asfiled. However, the most critical part of the removal of impurities andcareful monitoring of the fractions during ion-exchange columnpurification is documented. After the synthesis of TML from lysine, acareful collection of fractions was carried out and each fraction wasmonitored by thin layer chromatography, as reported in the parentapplication. The elution was done initially with deionized Milliporewater, followed by 0.5 M aqueous (aq.) ammonia (NH₃), followed by 1 Maq. ammonia. The fractions were visualized by ninhydrin test on the thinlayer chromatography (TLC) plate. The early and late eluting fractionswere found to be of impure or undesired impurity. The purest fractionsthus observed were then combined to yield TML of high purity a wasreported with no visible impurities by TLC (greater than 98%) andconform to correct mass spectral data and high-resolution proton NMRanalysis.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “derivative” means any of Formulas II-VI. Theinvention incorporates both TML and TML derivatives. As such, anymention of TML also encapsulates the TML derivative compounds.

The symbol “—” represents a covalent bond.

Reference to a chain, such as an alkyl, can mean either the branched orunbranched chain unless otherwise noted.

An “alkyl”, as used herein, means either a branched or unbranched alkylchain, and includes, for example, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and the like.

As used herein, “dilute” means ten percent or less in solution.

As used herein “excess” means a stoicheometry greater than 1:1.

A “mild base,” as used herein, means a dilute base.

The phrase “pharmaceutically acceptable” indicates that the designatedcarrier, vehicle, diluent, excipient(s), and/or salt must be chemicallyand/or physically compatible with the other ingredients comprising theFormulation, and physiologically compatible with the recipient thereof.

The invention provides a compound represented by Formula II:

wherein R′ is selected from an alkyl having between 1 and 5 carbon atomsand an aromatic ring.

The invention provides a compound represented by Formula III:

wherein R″ is selected from an alkyl having between 1 and 5 carbonatoms, and an aromatic ring.

The invention provides a compound represented by Formula IV:

wherein R′ is selected from an alkyl having between 1 and 5 carbonatoms, and CH3 and R″ is selected from an alkyl having between 1 and 5carbon atoms, and an aromatic ring.

The invention provides a compound represented by Formula V:

wherein a, a′, b, b′, c, c′, d, d′, e, and e′ are independently selectedfrom H, deuterium, and an alkyl having between 1 and 5 carbon atoms andR′ is selected from H, an alkyl having between 1 and 5 carbon atoms, oran aromatic ring.

In a preferred embodiment, each N is independently selected fromnitrogen or N15 labeled nitrogen.

Improved Method of Synthesis and Purification of TML and TML Derivatives

Starting Raw Material: L-lysine HCl (Sigma-Aldrich, St. Louis, Mo.),dimethylsulfate (99.99%) Sigma-Aldrich, St. Louis, Mo.), alkaline coppercarbonate (Sigma-Aldrich, St. Louis, Mo.), double distilled water(highly purified), Whatman 3MM Chromatography blotting paper (WhatmanInc., Florham Park, N.J.), sodium hydroxide (NaOH, 99%+) (Sigma-Aldrich,St. Louis, Mo.), Dowex SOWX8 ion exchange column (The Dow ChemicalCompany, Midland, Mich.).

L-lysine HCL (50 gm; 0.274 mol)) was dissolved in distilled water (500mL) and copper carbonate basic (72 gm; 0.326 mol) was added. The mixturewas boiled at 85 centigrade for 10 minutes.

The reaction mixture was cooled to room temperature and filtered withWhatman 3MM paper. The clear filtrate was mixed with dimethylsulfate,100 mL (1.055 mol) at room temperature, after which 325 mL of aq.sodiumhydroxide solution (10% aq.; 1.055 mol; w/v, in dd water) wasadded drop-wise during 30 minutes, then stirred at room temperature for60 min.

A 17″height×2″dia Dowex5OWx8 ion exchange column (H⁺ form) was washedwith de-ionized water prior to the addition of the TML solution.

The solution containing the TML was then added to Dowex5OWx8 column.

The column was copiously washed with 500 mL of distilled water. Thisprocess was repeated with 1700 mL water and 1000 mL and 700 mL fractionswere collected. The collection and monitoring of these fractions wereaccomplished by thing layer chromatography (TLC) and ninhydrin colortests.

Subsequently, 2M ammonium hydroxide solution was run and 8 fractions(each fraction 50 mL, followed by 100 mL) were collected. TLC analysiswas performed on all the fractions (ticsystem:MeOH:water:Aceticacid::80:10:10). These fractions are detailed inTable C below.

TABLE C Tabular Summary of Ion-Exchange Column Purification Steps.Fraction# Solvent Volume Result 1 H₂0 50 Negative 2 0.5M aq NH₃   75Negative 3 0.5M aq NH₃   75 Negative 4 0.5M aq NH₃   50 Negative 5 1M aqNH₃ 50 Positive 6 1M aq NH₃ 50 Positive 7 1M aq NH₃ 50 Positive 8 1M aqNH₃ 50 Positive 9 2M aq NH₃ 50 Positive 10 2M aq NH₃ 100 Positive 11 2Maq NH₃ 100 Positive 12 2M aq NH₃ 100 Positive 13 2M aq NH₃ 100 NegativeTable C Notes: 1. The Ion- Exchange purification on Dowex 50WX8 inchcolumn length 17 “heightX2” diameter. First elution (Fraction 1) 100 mlwater; subsequent elution in .5M aq NH_(3,) 200 ml; subsequent elutionin 1M aq NH_(3;) 200 ml; followed by last elution in 2M aq NH_(3;) 500ml. 2. TLC solvent system methanol:water:acetic acid:: 80:10:10 was usedto check all fractions after staining the TLC plate with ninhydrinsolution and observing colored stained band. 3. Fractions 5 to 10 werepure fractions, so they were combined and evaporated.

The eight fractions were combined and evaporated to yield an oil. Theoil was subsequently lyophilized at room temperature to yield a solid.

The solid was triturated in acetonitrile and filtered and washed withacetonitrile again.

The solid was dissolved in methanol/water (95:5::Methanol:ddWater) andfiltered with glass micro filter paper and the filtrate was evaporatedand lyophilized.

Large-Scale Purification of TML

A larger scale synthesis has been achieved, which is further amenable tolarge-scale production of TML. The larger scale synthesis of TMLincorporating step of final clean up to achieve purity of at least 98%or greater, and free of foreign materials, has been demonstrated. Theliterature procedure does not teach synthesis of high purity TML or TMLderivative, which could be applicable to pharmaceutical-grade product.Thus, the purification steps that allow larger scale synthesis can bedescribed as follows:

-   -   1. Running crude TML or TML derivative through an ion exchange        resin column;    -   2. Washing the ion exchange resin column with at least 4 times        the volume of water as the amount of present crude TML or TML        derivative;    -   3. Eluting the washed TML or TML derivative from the ion        exchange resin column;    -   4. Freezing the eluted solution and then lyophilize at room        temperature to prevent or minimize any decomposition of obtained        TML or TML derivative; and    -   5. Triturating the lyophilized solid TML or TML derivative.

In another embodiment, the following steps may include:

-   -   5. Dissolving the triturated TML or TML derivative into a polar        solvent;    -   6. Filtering the dissolved TML or TML derivative through a        microglass membrane filter; and    -   7. Lyophilizing the filtered TML or TML derivative at room        temperature.

This process is an improvement of what is disclosed in Frederic M. Vaz,Bela Melegh, Judit Bene, Dean Cuebas, Douglas A. Gage, Albert Bootsma,Peter Vreken, Albert H. Van Gennip, Loran L. Bieber And Ronald J. A.Wanders, Clin. Chem. 48:6, 826-834, 2002.

Quality Control

The following quality control parameters were obtained:

-   -   A. TLC' Samples 1 and 2 were purified TML made according to the        invention. Sample 3 was a reference TML purchased from        Sigma-Aldrich. The tic plates were Baker-flex silica gel 1B-F.        TLC Purity' was greater than 99%, and the spots were observed        after staining the spot with ninhydrin (10% in methnol) (FIG.        1).    -   B. 1H NMR: The 1H NMR (Proton in D₂0) was run on 300 MHz; 1.3932        ppm (methylene at C-2; 2H, broad singlet), 1.6643 ppm (methylene        at C-3 2H, broad, multiplet); 2.13 ppm (methylene at C-4; 2H,        broad singlet), 3.2928 ppm (methylene proton at C-5, 2H;        triplet); 3.2167 ppm (alpha H; 1H; triplet); 3.0798 ppm        (trimethyl H's; 9H) (FIGS. 2a and 2 b).    -   C. Mass Spectrum: Chemical Formula C₉H₂₀N 202, Molecular weight;        189.28. Four major fragmentation peaks were observed in positive        mode; m/e 189.3, m/e 211.2 (+Na ion), m/e 377.5 (possibly dimer        formation) and m/e 399.5 (possibly Na+ ion addition on dimmer)        (FIG. 3).

Salt Formation

The TML synthesized (as described above) had no external salt. Thecarboxylic group (which is negatively charged) and the trimethyl group(which is positively charged) form an internal salt. The alpha aminogroup picks up the proton from the ionized carboxylic group. Themolecular weight of this TML is 188.3 From our Mass Spectral analysis(positive ion) we get the molecular ion peak at 189.28 (One extra massin positive ion is proton adding from matrix). This data confirms MW of188.3.

The skilled artisan will understand that TML can exist as an externalsalt as well, such as a potassium salt.

1. A compound represented by Formula I

with a pharmaceutically acceptable purity level.
 2. The compound ofclaim 1, wherein said purity level is at least 98%.
 3. A compoundrepresented by Formula II

wherein R′ is selected from the group consisting of (an alkyl havingbetween 1 and 5 carbon atoms, and an aromatic ring).
 4. The compound ofclaim 3, wherein the compound is at least 98% pure.
 5. A compoundrepresented by Formula III

wherein R″ is selected from the group consisting of (an alkyl havingbetween 1 and 5 carbon atoms, CH3, and an aromatic ring).
 6. Thecompound of claim 5, wherein the compound is at least 98% pure.
 7. Acompound represented by Formula IV

wherein R′ is an alkyl having between 1 and 5 carbon atoms, or CH3, andwherein R″ is an alkyl having between 1 and 5 carbon atoms or anaromatic ring.
 8. The compound of claim 7, wherein the compound is atleast 98% pure.
 9. A compound represented by Formula V

wherein a, a′, b, b′; c, c′, d, d′, e, and e′ are independently selectedfrom the group consisting of (H, deuterium, and an alkyl having between1 and 5 carbon atoms); R′ is selected from the group consisting of (H,an alkyl having between 1 and 5 carbon atoms and an aromatic ring); andeach N is independently selected from nitrogen and N15 labeled nitrogen.10. The compound of claim 9, wherein the compound is at least 98% pure.11. A compound represented by Formula VI

wherein the a, b, b′, c, c′, d, d′, e, and e′ are independently selectedfrom the group consisting of (H, deuterium, and an alkyl having from 1to 5 carbon atoms), and each N is independently selected from nitrogenand N15 labeled nitrogen.
 12. The compound of claim 11, wherein thecompound is at least 98% pure.